Published May 1, 1966
Electrical Characteristics of Triturus
Egg Cells during Cleavage
SHIZUO ITO and NOBUAKI HORI
From the Cell PhysicsLaboratory, Department of Physiology,Columbia University, College
of Physiciansand Surgeons,New York, and the Department of Biology, Faculty of Science,
Kumamoto University,Kumamoto,Japan
Since Backman and Runnstr6m (1912) showed that the osmotic pressure of
Rana eggs diminishes at the time of fertilization, the permeabilities to water
and water-soluble substances of amphibian eggs have been studied under
various conditions (Krogh, Schmidt-Nielsen, and Zeuthen, 1938; Picken and
Rothschild, 1948; Holffreter, 1943; Kusa, 1951 ; de Luque and Hunter,
1959; de Luque, Hunter, and Hunter, 1961); and the permeabilities have
been correlated with activation processes (MaCho, 1959), and with oxygen
consumption (LCvtrup, 1962 a, 1962 b, and 1963).
Renewed interest in developmental aspects of cell permeability was stimulated by the work of K a n n o and Loewenstein (1963) who showed that the
m e m b r a n e potential and the specific resistance of amphibian egg cells increase progressively during development in the oocyte stages. T h e present
study concerns changes of this kind at later stages of development. It deals
with changes after fertilization, including those following egg cell cleavage.
It concerns in particular the m e m b r a n e resistance at the plane of cleavage,
which in preliminary experiments appeared to be smaller than that of the
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The Journal of General Physiology
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ABSTRACT The membrane potential in the blastomeres of dividing Triturus
egg cells increases progressively from the first cleavage to the late morula stages.
Both the animal and vegetal poles show the same increasing trend in potential;
there is no significant potential difference between them. Upon first cell cleavage, the total resistance of the egg cell surface in contact with the exterior decreases to about one-tenth of its value before cleavage, and then remains rather
constant up to the late morula stage. The specific resistance of this membrane
surface drops rather abruptly upon first cleavage, and rises progressively during
the morula stage. The resistance of the junctional membrane surface of the
blastomeres, that is, the membrane formed at the former planes of cleavage,
is small in relation to that of the cell surface in contact with the exterior. As a
result, the blastomeres are electrically coupled throughout all stages of embryonic development examined.
Published May 1, 1966
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rest of the cell surface (Ito, 1962 b). A f u r t h e r objective of the present study
was to e x a m i n e possible potential gradients along the egg polar axis d u r i n g
d e v e l o p m e n t , such as r e p o r t e d b y D o r f m a n (1934) a n d Flickinger a n d Blount
(1957).
MATERIALS
AND
METHODS
Fertilized eggs of the Japanese common newt (Trituruspyrrh0gaster) from the following
developmental stages were used: eggs before cleavage; 2-cell, 4-cell, 8-cell, 16-cell
stages; early morula, middle morula, and late morula. The jelly layer surrounding the
egg was removed with De Wecker scissors in Hohfreter's solution (1943); care wa.~
taken to avoid injury to the eggs. The chorion was left in place.
The general arrangement employed for electrical recording is illustrated in Fig. IA.
Two microelectrodes filled with 3 M KC1 were inserted into the egg. Electrode tip
y ~ p
B
"' p
FIGURE I. Diagrams of electrical setup. A, arrangement for measurements of membrane
resistance and voltage attenuation. For attenuation measurements the microelectrode
is moved to different positions within the egg or to different blastomeres. B, arrangement
for resistance measurements across the cleavage plane. Ch, chorion; M, egg cell membrane; P, potential recording; C, current recording. Preparation is in a physiological
salt solution.
resistances ranged from 20 to 50 Mohm. One of the electrodes was used for passing
rectangular pulses of current variable in magnitude and duration; and the other
electrode, for recording resting membrane potentials, and potential changes produced
by the current. The latter electrode was connected to a cathode ray tube through a
high impedance negative-capacity amplifier. The current was measured across a 100
kohm resistor in series with the current-passing circuit. The currents used were on the
order of 10-s A; their duration was 3 sec for the eggs before cleavage, and 0.5 to I sec
for the eggs after start of cleavage. One of the following two methods of recording was
used in most cases. In one, both the recording and the current-passing electrodes were
in the same blastomere (arrangement 1). In the other, the electrodes were in different
blastomeres (arrangement 2). In a few experiments, methods 1 and 2 were combined
by using three microelectrodes for simultaneous comparison of electrotonic potentials
in different blastomeres (see top inset of Fig. 5).
For determinations of the resistance across the cleavage plane, an arrangement of
four intracellular microelectrodes was used, as shown in Fig. IB. Current pulses of 100
msec duration and up to 10-6 A intensity were passed between two microelectrodes
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AIO0M_O.
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SItlZUO ITO AND NO~SUAraHORI MembraneResistance of Dividing Cell
I02I
of 10 tO 20 Mohm resistance, isolated from ground; and the resulting changes in membrane voltage recorded across the forming or already formed junctions of different
blastomeres. Current direction was alternated to minimize effects of polarization.
The present egg material was easily penetrated by the microelectrodes and offered
in this respect a more favorable material than echinoderm (Tyler, Monroy, Kao, and
Grundfest, 1956; Hiramoto, 1959) or Oryzias eggs (Ito and Ma6no, 1960).
RESULTS
Membrane Potentials W h e n the recording electrode established contact
with the chorion, a potential of 10 to 20 mv, negative with respect to the
external solution, was observed (Fig. 2 a). (Polarity of all potentials is expressed hereafter with respect to potential of external solution.) T h e potential
attained its m a x i m u m before passing through the chorion. After penetration
of the chorion, the potential reverted to zero. The magnitude of the potential
across the egg cell m e m b r a n e proper was, therefore, not affected by the
chorion contact potential. As the electrode advanced towards the egg cell
membrane, a progressively increasing positive potential developed (Fig. 2 b).
This potential was probably the result of mechanoelectric effects within the
electrode tip, due to mechanical strain.
U p o n penetration through the ooplasmic membrane, the potential suddenly changed polarity (Fig. 2 c). This coincided with the appearance of
electrotonic potentials, when arrangements 1 or 2 (see Methods) were used,
and gave assurance that the cell m e m b r a n e had been penetrated.
Table I summarizes the m e m b r a n e potentials so obtained with electrodes
located in the animal area and, from the 8-cell stage on, in both the animal
and vegetal areas. M e m b r a n e potentials increase progressively from 5 to 11
m v in the stages before cleavage, to values as high as 65 mv in the late morula
stage. Both the animal and the vegetal area show the same increasing trend
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YIo~
2. Potential changes
(downward, negative with respect
to outside) upon progressive electrode advancement through the
surface of an egg before cleavage.
See text.
Published May 1, 1966
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in potential. The average potential was somewhat lower at the vegetal area
than at the animal area; but the differences are not statistically significant.
Egg Surface t~sistance and Capacity F i g . 3 i l l u s t r a t e s a t y p i c a l c u r r e n t voltage relation in the fertilized egg before cleavage. At no instance, was a
TABLE
I
MEMBRANE
P O T E N T I A L S (MILLIVOLTS)* A T A N I M A L
AND VEGETAL POLES DURING DEVELOPMENT
Stages
1-cell
2-cell
4-cell
8-cell
16-cell
No. of eggs
examined
Vegetal area
No. of eggs
examined
5.64-2.8
15.74-6.5
20.34-7.1
22.64-4.5
31.14-9.2
35.34-9.5
41.14-9.1
53.84-10.1
24
15
21
10
21
28
13
15
21.3+3.3
28.34-9.6
33.44-9.3
37.84-11.1
48.64-13.5
12
19
27
13
14
* Mean values with standard deviation.
mv
40-
'~0
¢~IN
II
, • 5.3MG
20'
6
1
4
I
2
I
I
I
I
I
x 10-gA
20
.40
FIotWaz 3. Current-voltage relation of a fertilized egg before cleavage. Inward current,
downward; outward current, upward. Insets give examples of oscillograph recordings of
membrane current and voltage. Current pulse duration, 3 sec.
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early
Morula middle
late
Animal area
Published May 1, 1966
SHIzuo ITO AND NOBUAKIHoRI MembraneResistameof Dividing Cell
1o2 3
resistive voltage step found at the level of the chorion. The resistance of the
chorion seems to be negligible, and the current-voltage relations to reflect
entirely properties of the egg cell membrane. T h e slope gives the effective
surface resistances which ranged from 1.8 to 10 M o h m for outward, and 1.8
to 13 M o h m for inward currents.
Right after spawning, the effective surface resistance of the fertilized egg
averages 5 Mohm, which corresponds to a specific m e m b r a n e resistance of
630 kohm cm*. These resistances are in the same range as those of Bufo eggs
(Ma~no, 1959) and Oryzias eggs (Ito and Ma~no, 1960; Ito, 1962 a). With
further development, the total effective surface resistance undergoes changes
which are summarized in Fig. 4. Upon first cell cleavage, the effective re-
I
I
©
D
O
t
•
,I,
I
initiation
of
constriction
I
2
@
I
~11
4
S
Stage
41,
I
IG
t
•
I
middle
I
late
morula
FIOtrRE4. Total (effective)resistanceduring development. Mean values with standard
deviations. Distances on abscissa are not proportional to time.
sistance drops markedly and out of proportion with the increased surface
area. F r o m the 2-cell stage on, the effective resistance r e m a i v rather constant up to the morula stage, and rises then during this stage. During all this
time, the cell surface area increases continuously. The exact surface area during the later morula stage could not be determined. However, the trend of
the change in specific surface resistance is clear. This drops rather abruptly upon
first cleavage, and then increases progressively during the later morula stage.
T h e m e m b r a n e capacitance ranges from 0.7 to 1.0 # f / c m 2 and is rather
constant throughout development.
Resistance across the Cleavage Plane Fig. 5 illustrates the results of an experiment in which current is passed through one cell in the morula stage and
the resulting electrotonic voltages are recorded in two blastomeres 1 m m
apart. (See Fig. 5, top.) T h e most striking result is that the voltage attenu-
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5
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ates so little from one blastomere to another. In the example of Fig. 5 the
difference in voltage amounts to only 7°~ for outward current and 18°-/o for
inward current. Thus, the resistance must here reside largely in the cell surface in contact with the exterior; the resistances of the surfaces at the level of
the junction between blastomeres, i.e. the resistance across the former cleav-
A//A0o
/
I
I
I
I
I
I
I
6
4
2
2
4
6
8
I
X I O-eA
2O
#
0 x
4O
FmUR~ 5.
S i m u l t a n e o u s c u r r e n t - v o l t a g e relations in t w o b l a s t o m e r e s o f the m o r u l a
stage. Insets give examples of membrane current and voltages. Current pulse duration,
1 see.
age planes referred to as junctional membranes, are small by comparison. (For
a full experimental and theoretical analysis of a similar situation in a somatic
cell system, see Loewenstein and Kanno, 1964; and Loewenstein et al., 1965.)
T h e relatively low resistance across the junctional membranes is shown
particularly clearly by direct resistance measurements of the arrangement
shown diagrammatically in Fig. lB. A result is shown in Fig. 6. After completion of the first cell division, at a time when the junctional membranes
are fully formed, the resistance across the cleavage plane is on the average
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Published May 1, 1966
SttIZUO ITO AND NOBUAKIHogl MembraneResistance of Diviaing Cell
Io2 5
23 × 108 o h m as against 300 × 103 ohm at the nonjunctional surface m e m brane. An estimate of the specific resistance of the junctional membrane, obtained by treating the situation of current flow between blastomeres as in a
conducting cylinder, gives values of 550 o h m c m ~.
T h e above kinds of measurements were done only in the outermost layer
of blastomeres. No attempt was m a d e to record from more deeply located
blastomeres of the morula, since we feared that this might injure the membranes of the cells in the outer layer.
DISCUSSION
Values of m e m b r a n e potential are now available for several stages of egg cell
development. The measurements of K a n n o and Loewenstein (1963) cover a
T
i
o~
v
~
0:5
/
1
FmtrR~. 6. Membrane resistance
before (A) and after (B) first
cleavage.
wide growth span in oocytes; Ma~no's (1959) measurements encompass immature, mature, and activated eggs; and the present ones cover the span
from the end of fertilization to the late morula. Thus an attempt m a y now be
m a d e at piecing the various data together and giving a rough idea of the time
course of m e m b r a n e potential over a wide span of development. T h e data
come from different species; but these are phylogenetically close enough, and
the values overlap sufficiently to make such an attempt worthwhile, at least,
in providing an idea of the major trends. T h e attempt is m a d e in Fig. 7.
T h e resting potential increases progressively with growth in the oocytes
(Kanno and Loewenstein, 1963), reaches a peak in the immature egg, falls
rapidly to a low value in the mature egg (MaSno, 1959), to rise again progressively after the first cell division.
Dorfman (1934) has reported that a potential difference exists between
vegetal and animal poles in eggs of Rana temporaria and Rana arvalis. Dorfman's
observations, m a d e at a time when microelectrode techniques were not yet
developed, rely on measurements with electrodes of 20 to 30 # tip diameter.
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rnv
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These are too large to prevent injury and leakage artifacts. O u r measurements show no significant differences in potential between the poles.
Ashman, Kanno, and Loewenstein (1963) m a d e the first studies of electrical resistance across the cleavage plane of a dividing cell. T h e y observed a
progressive increase in resistance across this plane during the first cell division
in Asterias egg cells. In the early stages of cleavage, these authors found good
electrotonic coupling between the blastomeres; but towards the end of cleavage, when the junctional membranes are fully formed, the electrical coupling
diminished below detectable levels. Recent work by one of us on dividing
Echinarachnius egg cells confirms in all details this pattern of electrical coupling
(Ito and Loewenstein, 1965). T h e pattern in the present results on the clearI
I
I
I
Mature
I
I
J
I
i
T
0
,I
Oocyte
Immature
Fertilized 2-cell
I
Morula
Developmental stoge
FIotr~ 7. Diagram of changes in membrane potential during development of amphibian eggs. Curve drawn on the basis of data from oocyte stages of Xenopus (Kanno and
Loewenstein, 1963); from egg stagesof Bufo (Ma6no, 1959) and Triturus (present results).
ing Triturus egg is different. Here electrical coupling is detectable at all stages.
However, this does not necessarily mean that the permeability properties of
the junctional membranes are basically different from those in Asterias or
Echinarachnius. The difference in the pattern of electrical coupling, as revealed
by the method of measuring voltage attenuation, can reflect differences (a)
in resistivity of the junctional membranes; (b) in resistance of the nonjunctional m e m b r a n e surfaces; and (c) differences in shunting by the intercellular
medium. It is not possible, at present, to distinguish among the alternatives.
The close electrical coupling between cells at all embryonic stages examined is, however, of interest in itself. This opens the possibility that embryonic cells communicate through low junctional resistances. Communication of this sort has been shown to exist in a wide variety of adult epithelial
cells where molecules large enough to carry genetic information flow from
cell to cell (Loewenstein, 1966).
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Published May 1, 1966
Smzuo ITO AND NOmJAKIHORI MembraneResistanceof Dividing Cell
o~ 7
We are greatly indebted to ProfessorW. R. Loewenstein, Columbia University, for valuable discussion and criticismsin preparing the manuscript, and to Dr. H. Morita, Kyushu Univrsity, for
technical advice.
Receivedfor publication 2 September 1965.
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